Method for screening human intestinal Npt2B modulatory agents

ABSTRACT

A novel human sodium phosphate cotransporter expressed on the apical surface of intestinal epithelial cells (huNpt2B) and polypeptides related thereto, as well as nucleic acid compositions encoding the same, are provided. The subject polypeptides and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications. Also provided are methods of inhibiting Npt2B activity in a host and methods of treating disease conditions associated with Npt2B activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/052,664 filed Jan. 17, 2002, now U.S. Pat. No. 7,368,530,which is a divisional application of U.S. patent application Ser. No.09/499,964, filed Feb. 8, 2000, now U.S. Pat. No. 6,380,374, whichapplication claims priority to U.S. Provisional Patent Application No.60/119,321 filed Feb. 9, 1999, the disclosures of which are hereinincorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of the invention is ion transporters, particularly sodiumphosphate co-transporters.

2. Background of the Invention

Phosphorous plays an important role in membrane structure, transport andenergy storage. At normal physiological pH (e.g. pH of 7.4), inorganicphosphate (Pi) in plasma is made up of a 4:1 mixture of HPO₄ ²⁻ andH₂PO₄ ⁻. Of the 700 g of phosphorous present in the body, 0.1% ispresent in the extracellular fluid in a freely diffusible form. Theplasma level of Pi is maintained through control of Pi absorption in thesmall intestine, under the influence of vitamin D, and Pi excretion inthe kidney, under the influence of parathyroid hormone.

Absorption of Pi requires transepithelial transport. A critical step oftransepithelial transport of Pi is the uptake of Pi into epithelialcells. Pi uptake is accomplished by sodium phosphate co-transporterspresent on the apical surface of appropriate epithelial cells, e.g.intestinal and renal epithelial cells. A variety of sodium phosphateco-transporters have been identified to date, including: NaPi-1(rabbit); NPT1 (human); Npt1 (mouse); NaPi-2 (rat); NaPi-3 (human);NaPi-4 (opossum); NaPi-5 (flounder); NaPi-6 (rabbit); NaPi-7 (mouse);and NaPi of NBL-1 cells (bovine).

A variety of disease conditions are associated with disorders in Pimetabolism, where such disease conditions include those characterized bythe presence of hypophosphatemia, e.g. osteomalacia, hypocalciuria andrickets, and those characterized by the presence of hyperphosphatemia,e.g. hyperparathyroidism, hypocalcemia, vitamin D deficiency, softtissue or metastatic calcification, and the like. In particular,hyperphosphatemia is a characteristic of renal disease and failure, andis an underlying cause of many of the deleterious symptoms observed withsuch renal complications.

Methods of treating abnormalities in Pi metabolism are varied. Forexample, for disease conditions associated with the presence ofhypophosphatemia, treatment methodologies include: changes in diet toinclude phosphorous rich foods, supplementation with phosphorous salts,use of therapeutic agents, e.g. dipyridamole, and the like. For thosedisease conditions associated with hyperphosphatemia, treatment in theabsence of renal insufficiency may include hydration and/or the use ofaluminum based antacids that bind phosphorous in the intestinal lumen.Where renal insufficiency is present, phosphate binders and/or dietarymodification to restrict phosphorous intake are potentially useful, butdialysis is typically employed.

Because of the wide variety of disease conditions characterized by thepresence of abnormal Pi metabolism, there is continued interest in theidentification of the molecular components responsible for Pimetabolism. Of particular interest would be the identification of theintestinal transporter responsible for absorption and uptake of Pi inthe intestine.

RELEVANT LITERATURE

Hilfiker et al., Proc. Nat'l Acad. Sci. USA (1998) 95: 14564-14569discloses the mouse Npt2B nucleic acid sequence. Also of interest is:Feild et al., “Cloning and Characterization of a Sodium DependentPhosphate Transporter Isoform Expressed in Human Small Intestine andLung,” Published on Dec. 24, 1998 under GenBank Accession No. 4071357and submitted by Smithkline Beecham Pharmaceuticals, 709 Swedeland Road,King of Prussia, Pa. 19406 on Dec. 7, 1998). References disclosingsodium phosphate co-transporters include: Werner et al., Proc. Nat'lAcad. Sci. USA (1991) 88: 9608-9612; Chong et al., Genomics (1993) 18:355-359; Chong et al., Am. J. Physiol (1995) 268: F1038-F1045; Magagninet al., Proc. Nat'l Acad. Sci. USA (1993) 90: 5979-5983; Sorribas etal., J. Biol. Chem (1994) 269: 6615-6621; Werner et al., Am. J. Physiol(1995) 267: F311-F317; Verri et al., Am J. Physiol (1995) 268:F626-F633;Collins et al., FASEB J (1994) 8: 862-868; and Helps et al., Eur. J.Biochem. (1995) 228: 927-930.

Also of interest is WO 98/37198.

References providing background information on the role of sodiumphosphate co-transporters in phosphorous metabolism include: Tenenhouse,J. Bone Min. Res (1997) 12: 159; and Harrison's Principles of InternalMedicine, 14^(th) Ed. (1998) pp 2259-2263.

SUMMARY OF THE INVENTION

A novel human intestinal sodium phosphate co-transporter (i.e. Npt2B)and polypeptides related thereto, as well as nucleic acid compositionsencoding the same, are provided. The subject polypeptide and nucleicacid compositions find use in a variety of applications, includingresearch, diagnostic, and therapeutic agent screening applications, aswell as in treatment therapies. Also provided are methods of treatingdisease conditions associated with intestinal Npt2B function, e.g.conditions characterized by abnormal serum phosphate levels, such ashypo- and hyperphosphatemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the amino acid sequence of human Npt2B.

FIG. 2 provides the sequence of a nucleic acid encoding human Npt2B.

DETAILED DESCRIPTION OF THE INVENTION

A novel human intestinal sodium phosphate co-transporter (i.e. Npt2B)and polypeptides related thereto, as well as nucleic acid compositionsencoding the same, are provided. The subject polypeptide and/or nucleicacid compositions find use in a variety of different applications,including research, diagnostic, and therapeutic agentscreening/discovery/preparation applications. Also provided are methodsof treating disease conditions associated with intestinal Npt2Bfunction, e.g. conditions resulting in abnormal serum phosphate levels,such as hypo- and hyperphosphatemia.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Polypeptide Compositions

A novel human sodium phosphate co-transporter expressed in intestinalepithelial cells, as well as polypeptide compositions related thereto,are provided. The term polypeptide composition as used herein refers toboth the full length human protein as well as portions or fragmentsthereof. Also included in this term are variations of the naturallyoccurring human protein, where such variations are homologous orsubstantially similar to the naturally occurring protein, as describedin greater detail below. In the following description of the subjectinvention, the term Npt2B is used to refer to the wild type humanintestinal sodium phosphate co-transporter molecule of the subjectinvention.

The Npt2B protein of the subject invention is a membrane protein havinga number of transmembrane regions, where the number of putativetransmembrane regions based on the amino acid sequence is 10 (aspredicted using TMpred (www.Ch.embnet.org) based on TMbase(Tmbase-Adatabase of membrane spanning protein segments. Biol. Chem.Hoppe-Seyler 347, 166)). Npt2B is a type II sodium phosphateco-transporter. In its native environment, Npt2B is a co-transporter ofsodium cation and phosphate anion. Npt2B is expressed, among otherlocations, on the surface of intestinal epithelial cells, i.e. on theapical or intestinal luminal side of the epithelial cells, and thereforeprovides for the transport of sodium and phosphate ions from theintestinal lumen into the intestinal epithelial cells. Npt2b is alsoexpressed in lung type II alveolar cells where it may be involved intransport of phosphate into the cells to meet the increased demand formucin glycoprotein synthesis. Npt2B has a 23% amino acid sequenceidentity with human NPT1 as described in Chong et al., “Molecularcloning of the cDNA encoding a human renal sodium phosphate transportprotein and its assignment to chromosome 6p21.3-p23,” Genomics (1993)18: 355-359 and 52.5% identity to human Npt2a described in Maganin etal. “Expression Cloning of Human and Rat Renal Cortex Na/Pico-transport,” Proc. Natl. Acad. Sci. USA (1993) 90: 5979-5983 (asmeasured by MegAlign, DNAstar (1998) using clustal algorithm asdescribed in D. G. Higgins and P. M. Sharp. Fast and Sensitive multipleSequence Alignments on a Microcomputer (1989) CABIOS, 5: 151-153.Prameters used are ktuple 1, gpa penalty 3, window, 5 and diagonalssaved 5). Other Npt2B characteristics include: potential glycosylationsites on the extracellular loops.

Npt2B has an amino acid sequence as shown in FIG. 1 and identified asSEQ ID NO:01. Npt2B has a molecular weight based on its amino acidsequence of about 75 kDa, and more specifically 75598.52 dalton (asdetermined using Protean/DNAstar (1997) as per H. Nakashima et al. TheFolding Type of a pProtein is Related to the Amino Acid Composition. J.Biochem (Tokyo) 99: 153-162). The true molecular weight of Npt2B mayvary due to glycosylation and/or other postranslational modifications.As such, the actual molecular weight of Npt2B is likely to be in therange from about 70 to 130 kDa.

Npt2B homologs or proteins (or fragments thereof) that vary in sequencefrom the wild type sequence of the subject invention are also provided.By homolog is meant a protein having at least about 35%, usually atleast about 40% and more usually at least about 60% amino acid sequenceidentity to the Npt2B protein of the subject invention, (using MegAlign,DNAstar (1998) using clustal algorithm as described in D. G. Higgins andP. M. Sharp. Fast and Sensitive multiple Sequence Alignments on aMicrocomputer. (1989) CABIOS, 5: 151-153. Prameters used are ktuple 1,gpa penalty 3, windows 5 and diagonals saved 5).

Also provided are Npt2B proteins that are substantially identical to thehu Npt2B protein, where by substantially identical is meant that theprotein has an amino acid sequence identity to the sequence of Npt2B ofat least about 60%, usually at least about 65% and more usually at leastabout 70%.

The proteins of the subject invention are present in a non-naturallyoccurring environment, e.g. are separated from their naturally occurringenvironment. In certain embodiments, the subject proteins are present ina composition that is enriched for subject protein as compared to itsnaturally occurring environment. For example, purified Npt2B isprovided, where by purified is meant that Npt2B is present in acomposition that is substantially free of non-Npt2B proteins, where bysubstantially free is meant that less than 90%, usually less than 60%and more usually less than 50% of the composition is made up ofnon-Npt2B proteins. The proteins of the subject invention may also bepresent as an isolate, by which is meant that the protein issubstantially free of other proteins and other naturally occurringbiologic molecules, such as oligosaccharides, polynucleotides andfragments thereof, and the like, where the term “substantially free” inthis instance means that less than 70%, usually less than 60% and moreusually less than 50% of the composition containing the isolated proteinis some other naturally occurring biological molecule. In certainembodiments, the proteins are present in substantially pure form, whereby “substantially pure form” is meant at least 95%, usually at least 97%and more usually at least 99% pure.

In addition to the naturally occurring proteins, polypeptides which varyfrom the naturally occurring proteins are also provided, e.g. Npt2Bpolypeptides. By Npt2B polypeptide is meant an amino acid sequenceencoded by an open reading frame (ORF) of the gene encoding Npt2B,described in greater detail below, including the full length Npt2Bprotein and fragments thereof, particularly biologically activefragments and/or fragments corresponding to functional domains, e.g.transmembrane domain, and the like; and including fusions of the subjectpolypeptides to other proteins or parts thereof. Fragments of interestwill typically be at least about 10 aa in length, usually at least about50 aa in length, and may be as long as 300 aa in length or longer, butwill usually not exceed about 1000 aa in length, where the fragment willhave a stretch of amino acids that is identical to the subject proteinof at least about 10 aa, and usually at least about 15 aa, and in manyembodiments at least about 50 aa in length.

The subject proteins and polypeptides may be obtained from naturallyoccurring sources or synthetically produced. For example, Npt2B isgenerally derived from epithelial cells of the intestine, but may alsobe derived from other cell types or tissues in which expression of Npt2Bis identified, e.g. lung, etc. The subject proteins may also be derivedfrom synthetic means, e.g. by expressing a recombinant gene encodingprotein of interest in a suitable host, as described in greater detailbelow. Any convenient protein purification procedures may be employed,where suitable protein purification methodologies are described in Guideto Protein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may prepared from the original source, e.g. intestinalepithelial cells or the expression host, and purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,and the like.

Nucleic Acid Compositions

Also provided are nucleic acid compositions encoding Npt2B proteins orfragments thereof, as well as the Npt2B homologues of the presentinvention. By Npt2B nucleic acid composition is meant a compositioncomprising a sequence of DNA having an open reading frame that encodesNpt2B, i.e. a Npt2B gene, and is capable, under appropriate conditions,of being expressed as Npt2B. Also encompassed in this term are nucleicacids that are homologous or substantially similar or identical to thenucleic acids encoding Npt2B proteins. Thus, the subject inventionprovides genes encoding the human Npt2B of the subject invention andhomologs thereof. The human Npt2B gene has the nucleic acid sequenceshown in FIG. 2 and identified as SEQ ID NO:02, infra.

The source of homologous genes may be any species, e.g., primatespecies, particularly human; rodents, such as rats and mice, canines,felines, bovines, ovines, equines, yeast, nematodes, etc. Betweenmammalian species, e.g., human and mouse, homologs have substantialsequence similarity, e.g. at least 75% sequence identity, usually atleast 90%, more usually at least 95% between nucleotide sequences.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990), J. Mol. Biol. 215: 403-10 (using defaultsettings, i.e. parameters w=4 and T=17). The sequences provided hereinare essential for recognizing Npt2B-related and homologous proteins, andthe nucleic acids encoding the same, in database searches.

Nucleic acids encoding the Npt2B protein and Npt2B polypeptides of thesubject invention may be cDNA or genomic DNA or a fragment thereof. Theterm “Npt2B gene” shall be intended to mean the open reading frameencoding specific Npt2B proteins and polypeptides, and Npt2B introns, aswell as adjacent 5′ and 3′ non-coding nucleotide sequences involved inthe regulation of expression, up to about 20 kb beyond the codingregion, but possibly further in either direction. The gene may beintroduced into an appropriate vector for extrachromosomal maintenanceor for integration into a host genome.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 5′ and 3′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding a Npt2Bprotein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include 5′ and 3′ untranslatedregions found in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all ora part of the subject Npt2B protein. Double or single stranded fragmentsmay be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt.

The Npt2B genes are isolated and obtained in substantial purity,generally as other than an intact chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude a Npt2B sequence or fragment thereof, generally being at leastabout 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

Preparation of Npt2B Polypeptides

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the Npt2B polypeptides, asdescribed above. For expression, an expression cassette may be employed.The expression vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native tothe Npt2B gene, or may be derived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

Npt2B proteins and polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, HEK 293, CHO, Xenopus Oocytes, etc., may be used as theexpression host cells. In some situations, it is desirable to expressthe Npt2B gene in eukaryotic cells, where the Npt2B protein will benefitfrom native folding and post-translational modifications. Small peptidescan also be synthesized in the laboratory. Polypeptides that are subsetsof the complete Npt2B sequence may be used to identify and investigateparts of the protein important for function.

Uses of the Subject Npt2B Polypeptide and Nucleic Acid Compositions

The subject polypeptide and nucleic acid compositions find use in avariety of different applications, including research, diagnostic, andtherapeutic agent screening/discovery/preparation applications, as wellas in therapeutic compositions and methods employing the same.

Research Applications

The subject nucleic acid compositions find use in a variety of researchapplications. Research applications of interest include: theidentification of Npt2B homologs; as a source of novel promoterelements; the identification of Npt2B expression regulatory factors; asprobes and primers in hybridization applications, e.g. PCR; theidentification of expression patterns in biological specimens; thepreparation of cell or animal models for Npt2B function; the preparationof in vitro models for Npt2B function; etc.

Homologs of Npt2B are identified by any of a number of methods. Afragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate primers. Nucleic acids having sequencesimilarity are detected by hybridization under low stringencyconditions, for example, at 50° C. and 6×SSC (0.9 M sodium chloride/0.09M sodium citrate) and remain bound when subjected to washing at 55° C.in 1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate). Nucleic acids having a region of substantialidentity to the provided Npt2B sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the providedNpt2B sequences under stringent hybridization conditions. By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes.

The sequence of the 5′ flanking region may be utilized for promoterelements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where Npt2B is expressed. The tissuespecific expression is useful for determining the pattern of expression,and for providing promoters that mimic the native pattern of expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al. (1995), Mol. Med. 1:194-205;Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin and Richard-Foy(1995), Eur. J. Biochem. 232:620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of Npt2B geneexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans-acting factors thatregulate or mediate Npt2B gene expression. Such transcription ortranslational control regions may be operably linked to a Npt2B gene inorder to promote expression of wild type or altered Npt2B or otherproteins of interest in cultured cells, or in embryonic, fetal or adulttissues, and for gene therapy.

Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in geometric amplification reactions, suchas geometric PCR, a pair of primers will be used. The exact compositionof the primer sequences is not critical to the invention, but for mostapplications the primers will hybridize to the subject sequence understringent conditions, as known in the art. It is preferable to choose apair of primers that will generate an amplification product of at leastabout 50 nt, preferably at least about 100 nt. Algorithms for theselection of primer sequences are generally known, and are available incommercial software packages. Amplification primers hybridize tocomplementary strands of DNA, and will prime towards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of Npt2B gene expression in the sample.

The sequence of a Npt2B gene, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions,deletions, or a combination thereof. Deletions may further includelarger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLAGsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for site specific mutagenesis may be found in Gustin et al.(1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicelli etal. (1985), Mol. Gen. Genet. 199:537-9; and Prentki et al. (1984), Gene29:303-13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3-15.108; Weiner et al. (1993), Gene 126:35-41; Sayers et al.(1992), Biotechniques 13:592-6; Jones and Winistorfer (1992),Biotechniques 12:528-30; Barton et al. (1990), Nucleic Acids Res18:7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67-70; and Zhu(1989), Anal Biochem 177:120-4. Such mutated genes may be used to studystructure-function relationships of Npt2B, or to alter properties of theprotein that affect its function or regulation.

The subject nucleic acids can be used to generate transgenic, non-humananimals or site specific gene modifications in cell lines. Transgenicanimals may be made through homologous recombination, where theendogenous Npt2B locus is altered. Alternatively, a nucleic acidconstruct is randomly integrated into the genome. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like.

The modified cells or animals are useful in the study of Npt2B functionand regulation. Of interest is the use of Npt2B genes to constructtransgenic animal models of Npt2B related disease conditions, e.g.hyper- or hypophosphatemia. Thus, transgenic animal models of thesubject invention include endogenous Npt2B knockouts in which expressionof endogenous Npt2B is at least reduced if not eliminated, where suchanimals also typically express an Npt2B peptide of the subjectinvention, e.g. the Npt2B protein of the subject invention or a fragmentthereof. Where a nucleic acid having a sequence found in the human Npt2Bgene is introduced, the introduced nucleic acid may be either a completeor partial sequence of the Npt2B gene. A detectable marker, such as lacZ may be introduced into the Npt2B locus, where upregulation of Npt2Bexpression will result in an easily detected change in phenotype. Onemay also provide for expression of the Npt2B gene or variants thereof incells or tissues where it is not normally expressed, at levels notnormally present in such cells or tissues.

DNA constructs for homologous recombination will comprise at least aportion of the Npt2B gene of the subject invention, wherein the gene hasthe desired genetic modification(s), and includes regions of homology tothe target locus. DNA constructs for random integration need not includeregions of homology to mediate recombination. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol.185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on Npt2B activity.

Diagnostic Applications

Also provided are methods of diagnosing disease states based on observedlevels of Npt2B or the expression level of the Npt2B gene in abiological sample of interest. Samples, as used herein, includebiological fluids such as blood, cerebrospinal fluid, tears, saliva,lymph, dialysis fluid, semen and the like; organ or tissue culturederived fluids; and fluids extracted from physiological tissues. Alsoincluded in the term are derivatives and fractions of such fluids. Thecells may be dissociated, in the case of solid tissues, or tissuesections may be analyzed. Alternatively a lysate of the cells may beprepared.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. Diagnosis may be performedby a number of methods to determine the absence or presence or alteredamounts of normal or abnormal Npt2B in a patient sample. For example,detection may utilize staining of cells or histological sections withlabeled antibodies, performed in accordance with conventional methods.Cells are permeabilized to stain cytoplasmic molecules. The antibodiesof interest are added to the cell sample, and incubated for a period oftime sufficient to allow binding to the epitope, usually at least about10 minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent.Alternatively, the secondary antibody conjugated to a fluorescentcompound, e.g. fluorescein, rhodamine, Texas red, etc. Final detectionuses a substrate that undergoes a color change in the presence of theperoxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Alternatively, one may focus on the expression of Npt2B. Biochemicalstudies may be performed to determine whether a sequence polymorphism ina Npt2B coding region or control regions is associated with disease.Disease associated polymorphisms may include deletion or truncation ofthe gene, mutations that alter expression level, that affect theactivity of the protein, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of Npt2B can be compared to expression levels of the normalallele by various methods known in the art. Methods for determiningpromoter or enhancer strength include quantitation of the expressednatural protein; insertion of the variant control element into a vectorwith a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that express Npt2Bmay be used as a source of mRNA, which may be assayed directly orreverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985),Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890; andDelahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type Npt2B gene sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilized on asolid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

Screening for mutations in Npt2B may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inNpt2B proteins may be used in screening. Where many diverse geneticmutations lead to a particular disease phenotype, functional proteinassays have proven to be effective screening tools. The activity of theencoded Npt2B protein may be determined by comparison with the wild-typeprotein.

Diagnostic methods of the subject invention in which the level of Npt2Bgene expression is of interest will typically involve comparison of theNpt2B nucleic acid abundance of a sample of interest with that of acontrol value to determine any relative differences, where thedifference may be measured qualitatively and/or quantitatively, whichdifferences are then related to the presence or absence of an abnormalNpt2B gene expression pattern. A variety of different methods fordetermine the nucleic acid abundance in a sample are known to those ofskill in the art, where particular methods of interest include thosedescribed in: Pietu et al., Genome Res (June 1996) δ: 492-503; Zhao etal., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin. Biotechnol.(October 1997) δ: 542-546; Raval, J. Pharmacol Toxicol Methods (November1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216:299-304; Stolz & Tuan, Mol. Biotechnol (December 19960 6:225-230; Honget al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem(1984) 143:298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

Screening Assays

The subject Npt2B polypeptides find use in various screening assaysdesigned to identify therapeutic agents. Thus, one can use a cell modelsuch as a host cell, e.g. CHO, HEK293, COS7, Xenopus Oocyte, etc., whichhas been transfected in a manner sufficient to express Npt2B on itssurface. One can then contact the cell with a medium comprising sodiumand phosphate ions, and measure the amount of phosphate anions that areinternalized in the cell, where measurements are taken in both controlenvironments and test environments, e.g. in the presence of a candidateNpt2B modulator compound, e.g. an Npt2B agonist or an Npt2B antagonistor inhibitor. To assist in detection of Pi uptake, labeled phosphorousis present in the medium, where any convenient label may be employed,such as an isotopic label, e.g. as present in ³²P or ³³P. Alternatively,current measurements may be taking using well known electrophysiologicalmethods (see e.g. Electrophysiology, A practical Approach (IRLPress)(1993)), from which the uptake of Pi may be determined. Examplesof assays for measuring Pi uptake are provided in: Maganin et al., Proc.Nat'l Acad. Sci. USA (July 1993) 90: 5979-5983; and Helps et al., Eur.J. Biochem (1995) 228: 927-930.

Also of interest in screening assays are non-human transgenic animalswhich express functional Npt2B, where such animals are described above.In many embodiments, the animals lack endogenous Npt2B. In using suchanimals for screening applications, a test compound(s) is administeredto the animal, and the resultant changes in phenotype, e.g. serum Pilevel, if any, are compared with a control.

Alternatively, in vitro models may be prepared and employed. Forexample, Npt2B binding activity may be measured in an in vitroenvironment in which binding events between Npt2B and candidate Npt2Bmodulatory agents are monitored. In yet other in vitro models, syntheticlipid bilayers incorporating the subject Npt2B cotransporter areprepared, an Pi passage from one side of the lipid bilayer to another ismeasured. Examples of such synthetic lipid bilayer assays can be foundin: Brutyan et al., Biochimica et Biophysica Acta (1995) 1236:339-344;Wonderlin et al., Biophys. J (1990) 58:289-297; and Suarez-Isla et al.Biochemistry (1983) 22:2319-2323.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used.

A variety of different candidate therapeutic agents that serve as eitherNpt2B agonists or antagonists may be screened by the above methods.Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Of particular interest in many embodiments are screening methods thatidentify agents that selectively modulate, e.g. inhibit, the subjectNpt2B and not other sodium phosphate cotransporters, such as the renalsodium phosphate cotransporters, e.g. Npt1. To identify such agents, amulti step screening protocol can be performed, where agents thatinhibit Npt2B in a first assay are then assayed for their ability toinhibit non-Npt2B transporters. Any convenient assay can be employed inthis second step assay, such as those disclosed in Maganin et al., Proc.Nat'l Acad. Sci. USA (July 1993) 90: 5979-5983; and Helps et al., Eur.J. Biochem (1995) 228: 927-930.

Npt2B Nucleic Acid and Polypeptide Therapeutic Compositions

The nucleic acid compositions of the subject invention also find use astherapeutic agents in situations where one wishes to enhance Npt2Bactivity in a host, e.g. disease conditions associate withhypophosphatemia. The Npt2B genes, gene fragments, or the encoded Npt2Bprotein or protein fragments are useful in gene therapy to treatdisorders associated with Npt2B defects. Expression vectors may be usedto introduce the Npt2B gene into a cell. Such vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and thelike, where the vectors are able to transiently or stably be maintainedin the cells, usually for a period of at least about one day, moreusually for a period of at least about several days to several weeks.

The gene or Npt2B protein may be introduced into tissues or host cellsby any number of routes, including viral infection, microinjection, orfusion of vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells.

Methods of Modulating Npt2B Activity

The subject invention provides methods of modulating Npt2B activity in acell, including methods of increasing Npt2B activity (e.g. methods ofenhancing Pi transport), as well as methods of reducing or inhibitingNpt2B activity, e.g. methods of stopping or limiting Pi transport. Insuch methods, an effective amount of an Npt2B modulatory agent iscontacted with the cell.

Also provided are methods of modulating, including enhancing andinhibiting, Npt2B activity in a host. In such methods, an effectiveamount of active agent that modulates the activity of Npt2B in vivo,e.g. usually enhances or inhibits Npt2B activity, is administered to thehost. The active agent may be a variety of different compounds,including a naturally occurring or synthetic small molecule compound, anantibody, fragment or derivative thereof, an antisense composition, andthe like.

Of particular interest in certain embodiments are agents that reduceNpt2B activity, e.g. Pi transport, by at least about 10 fold, usually atleast about 20 fold and more usually at least about 25 fold, as measureby the Oocyte Transport Assay as described in Magagnin, supra. In manyembodiments, of particular interest is the use of compounds that reduceNpt2b activity by at least 100 fold, as compared to a control.

Also of interest is the use of agents that, while providing for reducedNpt2B activity, do not substantially reduce the activity of other sodiumphosphate co-transporters, e.g. Npt1, etc., if at all. Thus, the agentsin this embodiment are selective inhibitors of Npt2B. An agent isconsidered to be selective if it provides for the above reduced Npt2Bactivity, but substantially no reduced activity of at least one othertype of sodium phosphate co-transporter, where substantially no meansless than 10 fold reduction, usually less than 5 fold reduction and inmany instances less than 1 fold reduction, where activity is measured asdescribed in Magagnin, supra.

Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Also of interest as active agent are antibodies that at least reduce, ifnot inhibit, the target Npt2B activity in the host. Suitable antibodiesare obtained by immunizing a host animal with peptides comprising all ora portion of the target protein, e.g. Npt2B. Suitable host animalsinclude mouse, rat sheep, goat, hamster, rabbit, etc. The origin of theprotein immunogen may be mouse, human, rat, monkey etc. The host animalwill generally be a different species than the immunogen, e.g. humanNpt2B used to immunize mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part ofNpt2B, where these residues contain the post-translation modifications,such as glycosylation, found on the native target protein. Immunogenscomprising the extracellular domain are produced in a variety of waysknown in the art, e.g. expression of cloned genes using conventionalrecombinant methods, isolation from HEC, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using Npt2B bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

For in vivo use, particularly for injection into humans, it is desirableto decrease the antigenicity of the antibody. An immune response of arecipient against the blocking agent will potentially decrease theperiod of time that the therapy is effective. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036). Alternatively, the antibody of interest maybe engineered by recombinant DNA techniques to substitute the CH1, CH2,CH3, hinge domains, and/or the framework domain with the correspondinghuman sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

In yet other embodiments of the invention, the active agent is an agentthat modulates, and generally decreases or down regulates, theexpression of the gene encoding the target protein in the host. Forexample, antisense molecules can be used to down-regulate expression ofNpt2B in cells. The anti-sense reagent may be antisense oligonucleotides(ODN), particularly synthetic ODN having chemical modifications fromnative nucleic acids, or nucleic acid constructs that express suchanti-sense molecules as RNA. The antisense sequence is complementary tothe mRNA of the targeted gene, and inhibits expression of the targetedgene products. Antisense molecules inhibit gene expression throughvarious mechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′—NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. Generally, the desired result isat least an enhancement or reduction in phosphate anion intestinalabsorption, as measured by plasma Pi levels, as compared to a control.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredmodulation of Npt2B activity, e.g. desired reduction in Pi absorptionand plasma Pi levels. Thus, the agent can be incorporated into a varietyof formulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, e.g. antisense composition, it may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the therapeutic DNA, then bombarded into skin cells.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

The subject methods find use in the treatment of a variety of differentdisease conditions involving Npt2B activity. As such, the diseaseconditions treatable according to the subject methods include diseasescharacterized by abnormally high Pi absorption and disease conditionscharacterized by abnormally low Pi absorption. Disease conditionsresulting from abnormally low Npt2B activity are those characterized bythe presence of hypophosphatemia, and include: osteomalacia,hypocalciurea, rickets, and the like. Disease conditions resulting fromabnormally high Npt2B activity are those characterized by the presenceof hyperphosphatemia and include: hyperparathyroidism, hypocalcemia,vitamin D deficiency, soft tissue or metastatic calcification, and thelike. Of particular interest is the use of the subject methods to treathyperphosphatemia resulting from renal insufficiency, e.g. caused byrenal disease resulting in at least impaired renal function, and thelike.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as hyperphosphatemia. Assuch, treatment also includes situations where the pathologicalcondition, or at least symptoms associated therewith, are completelyinhibited, e.g. prevented from happening, or stopped, e.g. terminated,such that the host no longer suffers from the pathological condition, orat least the symptoms that characterize the pathological condition.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

Kits with unit doses of the active agent, usually in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

EXPERIMENTAL A. Identification of the Npt2B Sequence

Comparison of type II sodium-phosphate cotransporter protein sequencesfrom different species available from public databases revealed thatwhilst most were very closely related, the bovine and flounder sequencesappeared to form a distinct sub-family. The Incyte LifeSeq database wasthus searched for Npt2-like clones that more closely resembled thebovine sequence than they did the human. A number of clones wereidentified and three of them were obtained and the DNA sequence of theentire inserts determined. DNA sequencing was performed on an automatedsequencer (PE/Applied Biosystems Model 373A, Foster City, Calif.) usingvendor's dye dideoxy termination sequencing kit. Comparison of thesequences revealed that they represented the same cDNA and that thelongest was only a partial clone missing approximately 150 amino acidsfrom the N-terminus, based on homology to the bovine protein. Theconsensus sequence was used to further screen the LifeSeq database and alarge number of clones were identified, including one which appeared tocontain the full-length coding sequence. The latter was obtained fromIncyte and sequenced. This revealed the presence of a 689 amino acidopen reading frame which appeared to be a human member of thebovine/flounder type II cotransporter subfamily. The majority of theclones identified in the LifeSeq database were from libraries derivedfrom lung-related tissue samples, however some of the clones were fromlibraries of small intestine and ovarian origin. This suggested thatthis cDNA might be a candidate for the human intestinal sodium-phosphatecotransporter. Experiments using RT-PCR confirmed the expression of thisgene in cDNA derived from human small intestine samples (obtained fromClontech Corporation, Palo Alto, Calif.). Subsequently, assignment ofthis sequence as the human intestinal transporter was strengthened by ahigh degree of homology to published sequences for Xenopus (A.Ishizuya-Oka et al. (1997) Temporal and Spatial Expression of anIntestinal Na⁺/PO₄ ³⁻Cotransporter Correlates With EpithelialTransformation During Thyroid Hormone-Dependent Frog Metamorphosis.Development Genetics 20:53-66) and mouse (H. Hilfiker et al.,Characterization of a murine type II sodium-phosphate cotransporterexpressed in mammalian small intestine. PNAS 1998 95:14564-14569)intestinal transporters.

B. Expression in Mammalian Cells and Assay Protocol for Na/PiTransporter

The Human Npt2B cDNA is cloned into a suitable constitutive mammalianexpression vector such as pcDNA3.1 or pREP9 (both from Invitrogen,Carlsbad, Calif.). If required, the cDNA is cloned into an induciblevector such as pLK-neo, a glucocorticoid-inducible vector (from Prof.Nicholas Fasel, Institute of Biochemistry, University of Lausanne,Switzerland; as described in Gene, 111, 199-206 (1992)). A number ofmammalian cell lines, e.g. HEK 293, CHO, MDCK, BHK and NIH 3T3 aretransfected with the expression constructs, and screened for stableexpression of the recombinant transporter using a version of the assaydescribed below.

Cells expressing the transporter are plated in 96 well tissue cultureplates at 50,000 to 100,000 cells per well in 200 μl of suitable mediaand allowed to adhere for 3-16 hours. Immediately before the assay,cells are washed three times with sodium- and phosphate-free wash buffer(137 mM N-methyl-D-glucamine, 5.4 mM KCl, 2.8 mM CaCl₂, 1.2 mM MgCl₂, 10mM HEPES, pH adjusted to 7.4 with HCl). The following components arethen added, (1) 50 μl of reaction buffer (137 mM NaCl, 5.4 mM KCl, 2.8mM CaCl₂, 1.2 mM MgCl₂, 0.1 mM kH₂PO₄, 10 mM HEPES, pH adjusted to 7.4with KOH), (2) 40 μl of test compounds diluted in reaction buffer and(3) 10 μl of a 10× phosphate solution (final concentration of phosphateis 100 μM) containing either ³²P (0.25 μCi) or ³³P (0.5 μCi) as tracer.Plates are incubated at room temperature for 20-30 minutes, and thereaction is stopped by removing the uptake solution and washing thecells 3 times with ice-cold stop solution (137 mM NaCl, 10 mM Tris-HCl,pH 7.2). The radioactivity taken up by the cells is determined bycounting after the addition of 150 μl of scintillation cocktail.Controls consist of cells incubated without test compound (vehiclealone) and cells incubated with N-methyl-D-glucamine in the reactionbuffer instead of sodium. Inhibitory activity is expressed as percentageinhibition of sodium-dependent uptake of the tracer. Using the aboveprotocol, candidate therapeutic agents are screened for their Npt2Bmodulatory activity.

It is apparent from the above results and discussion that a novel humanintestinal sodium phosphate cotransporter, as well as polypeptidesrelated thereto and nucleic acid compositions encoding the same, areprovided by the subject invention. These polypeptide and nucleic acidcompositions find use in a variety of diverse applications, includingresearch, diagnostic, screening and therapeutic applications. Alsoprovided are novel methods of treating diseases associated abnormalitiesin plasma phosphate levels, as the identification of the subject sodiumphosphate cotransporter provides for an additional target fortherapeutic agents for such diseases. Accordingly, the subject inventionprovides for a significant contribution to the field.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method of screening to identify Npt2B modulatory agents, saidmethod comprising: contacting a cell expressing a functional Npt2Bpolypeptide on its surface, said polypeptide comprising the amino acidsequence set forth in SEQ ID NO:1 on its surface with a candidate agentin the presence of phosphorous anion; and determining the amount ofphosphorous anion uptake by said cell wherein an altered uptake in theamount of phosphorous agent in the presence of a candidate agentidentifies an Npt2B modulatory agent.
 2. The method according to claim1, wherein said phosphorous anion is labeled with a detectable label. 3.The method according to claim 2, wherein said label is isotopic.